Silencing Ditylenchus destructor cathepsin L-like cysteine protease has negative pleiotropic effect on nematode ontogenesis

Ditylenchus destructor is a migratory plant-parasitic nematode that severely harms many agriculturally important crops. The control of this pest is difficult, thus efficient strategies for its management in agricultural production are urgently required. Cathepsin L-like cysteine protease (CPL) is one important protease that has been shown to participate in various physiological and pathological processes. Here we decided to characterize the CPL gene (Dd-cpl-1) from D. destructor. Analysis of Dd-cpl-1 gene showed that Dd-cpl-1 gene contains a signal peptide, an I29 inhibitor domain with ERFNIN and GNFD motifs, and a peptidase C1 domain with four conserved active residues, showing evolutionary conservation with other nematode CPLs. RT-qPCR revealed that Dd-cpl-1 gene displayed high expression in third-stage juveniles (J3s) and female adults. In situ hybridization analysis demonstrated that Dd-cpl-1 was expressed in the digestive system and reproductive organs. Silencing Dd-cpl-1 in 1-cell stage eggs of D. destructor by RNAi resulted in a severely delay in development or even in abortive morphogenesis during embryogenesis. The RNAi-mediated silencing of Dd-cpl-1 in J2s and J3s resulted in a developmental arrest phenotype in J3 stage. In addition, silencing Dd-cpl-1 gene expression in female adults led to a 57.43% decrease in egg production. Finally, Dd-cpl-1 RNAi-treated nematodes showed a significant reduction in host colonization and infection. Overall, our results indicate that Dd-CPL-1 plays multiple roles in D. destructor ontogenesis and could serve as a new potential target for controlling D. destructor.


Developmental stage expression patterns of Dd-cpl-1
The expression levels of Dd-cpl-1 in different developmental stages were determined by RT-qPCR .All samples including approximately 10,000 eggs, 5000 J2s, 500 J3s, 500 J4s, 200 female adults and 200 male adults were collected.Total RNA from each sample was extracted separately using TranZol™ Up Plus RNA Kit (Cat# ER501, TransGen Biotech, Beijing, China) and analyzed with a Nanodrop 2000 spectrometer (Thermo Fisher Scientific, Waltham MA, USA).First-strand complementary DNA (cDNA) was synthesized individually using the Prime-Script™ RT reagent Kit with gDNA Eraser (Cat# RR047Q, Takara, Dalian, China).The alpha-tubulin (tba-1) and actin (act-1) genes from D. destructor were used as internal reference genes 10 .The primers are listed in Table 1.The tba-1 and act-1 genes have been widely used as reference genes for RT-qPCR in various plant-parasitic nematodes since they are expressed constitutively in most cells and tissues.RT-qPCR was performed using the SYBR® Green Realtime PCR Master Mix (Cat# QPK-201 T, Toyobo, Osaka, Japan) and an ABI QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific, Waltham MA, USA).The relative expression level of Dd-CPL-1 was calculated by the 2 −ΔΔCt method 27 .These experiments were conducted with three independent replicates.

Complementary DNA cloning of Dd-cpl-1
The total RNA from mixed-stage nematodes was extracted, and the cDNA was synthesized as described above.The encoding sequence of Dd-cpl-1 was cloned from cDNA using Ex Taq® DNA Polymerase (Cat# RR001Q, Takara, Dalian, China) with a pair of specific primers: Ddcpl-cF and Ddcpl-cR (Table 1).The amplified products were purified and ligated to the pMD TM  www.nature.com/scientificreports/

In situ hybridization of Dd-cpl-1
To determine the localization of Dd-cpl-1, in situ hybridization was performed in different developmental stages of D. destructor.Specific sense (ISH-S) and antisense (ISH-A) primers (Table 1) were designed to amplify a 343 bp fragment based on the cloned PCR product of Dd-cpl-1.The purified PCR fragment was used as template to synthesize the digoxigenin (DIG)-labeled sense and antisense RNA probes using a DIG-labelling Kit (Cat# DDLK-010, MyLab, Beijing, China).In situ hybridization was performed as previously described 28 following the manufacturer's protocol of the DIG hybridization detection Kit (Cat# DIGD-120, MyLab, Beijing, China).
Final observations and photographs of nematode specimens were made using an 80i optical microscope (Nikon, Tokyo, Japan).

Double-stranded RNA (dsRNA) synthesis, RNA interference and phenotype analysis
The dsRNA fragments for Dd-cpl-1 and the green fluorescence protein (GFP) gene were prepared in vitro using T7 RNAi Transcription Kit (Cat# TR102-01, Vazyme, Nanjing, China) and the primers listed in Table 1.The quality and concentration of dsRNA were estimated by gel electrophoresis and Nanodrop 2000 (Thermo Fisher Scientific, Waltham MA, USA).
To investigate the effect of Dd-cpl-1 silencing on embryonic development, RNAi was performed on 1-cell stage eggs.Approximately 2000 eggs were washed with DEPC water, from which 30 1-cell stage eggs were picked and placed in 96-well plates (Scoperta Life Sciences, Wayne, USA) (one egg per well), followed by adding 50 μg dsRNA in 50 μL.The plates were maintained at 25 °C in a dark incubator with 70% relative humidity.Development of these treated eggs was observed and recorded every 6 h under an IX71 inverted microscope (Olympus, Tokyo, Japan) till hatching.The hatching rates (number of hatched eggs/total number of eggs) were calculated.The rest of the washed eggs were soaked into dsRNA (final concentration 1 μg/μL) for 24 h, and used for qPCR analysis to check the RNAi efficiency.
The effect of RNAi during post-embryonic development was also analyzed by soaking juvenile nematodes into dsRNA.Several thousands of synchronized J2s, J3s, J4s respectively, were washed with DEPC water and soaked into dsRNA (final concentration 1 μg/μL) soaking solution 10 (50 mM octopamine hydrochloride, 3 mM spermidine and 0.01% TritonX-100) (Sigma-Aldrich, St Louis, MO, USA) for 24 h in a dark rotator (10 rpm) at 25 °C.Then, these dsRNA-treated nematodes at the different juvenile stages were separately inoculated onto small slices (1 × 1 × 0.5 cm) of sweet potato roots (approximately 200 individuals each) and cultured at 25 °C in the dark incubator with 70% relative humidity.The proportion and body size (length and width) of nematodes from one developmental stage to the next stage were observed and recorded at the specific time according to the developmental timeline of untreated nematodes (J2-J3-J4-adults: 60-48-84 h).The rest of the soaked juveniles were retained for checking the RNAi efficiency by qPCR analysis.
To analyze the potential effects of Dd-cpl-1 RNAi on the nematode reproduction, the quantity and quality of eggs laid by dsRNA-treated females were analyzed.Male and female J4s were collected and cultured separately onto two different sweet potato root slices for 84 h to get virgin adult nematodes.Approximately 200 virgin female adults were collected and incubated in the dsRNA (final concentration 1 μg/μL) soaking solution for 24 h.The mating assay was performed as described 29 with some modifications.Briefly, after soaking, 30 of the soaked female adults were transferred into 96-well plates (Scoperta Life Sciences, Wayne, USA) (one female per well), followed by adding 100 μL sterile water.Non-RNAi treated virgin male adults were added into these plates (one male per well), and the plates were stood upright to allow that males and females could easily contact each other inducing a successful mating.The number and hatching rates of laid eggs were recorded every 24 h under an IX71 inverted microscope (Olympus, Tokyo, Japan).The rest of the soaked female adults were retained for qPCR analysis to check the RNAi efficiency.
To detect the effect of Dd-cpl-1 RNAi silencing on the infectivity of D. destructor, the number of colonizing nematodes and infection area of dsRNA-treated nematodes in sweet potatoes were analyzed.Mixed-stage www.nature.com/scientificreports/nematodes treated with dsRNA soaking solution as mentioned above were inoculated in sweet potato roots (approximately 500 nematodes each).The inoculated roots were then maintained at 25 °C in a dark incubator with 70% relative humidity.After 25 days, the nematodes were collected from the inoculated roots as described before, and counted.The infection area was photographed using a Canon EOS RP digital camera (Canon, Tokyo, Japan) and measured via ImageJ (https:// imagej.net/ softw are/ imagej/).All pictures from each biological replicate were taken under the same conditions and the infection area was measured by using the ImageJ software.The results were normalized as the percentage value relative to the dsGFP control.
The same quality and quantity of nematodes treated with dsGFP were used as control.All experiments were performed at least in triplicate with three biological replicates.

Sequence and phylogenetic analysis of Dd-cpl-1
The Dd-cpl-1 gene sequence was obtained based on our previously published genome data of D. destructor 26 .The nucleotide sequence of Dd-cpl-1 (Gene ID in WormBase ParaSite Database: Dd_06582) spans 2841 bp on D. destructor genome.This gene has only one transcript composing of ten exons and nine introns (Fig. 1a).Dd-cpl-1 mRNA encompasses 16 bp of the 5′ untranslated region (UTR) and 149 bp of the 3′ UTR, and has an ORF of 1131 bp encoding a protein of 376 amino acids (aa) with a 20 aa signal peptide.Dd-CPL-1 N-terminus contains a I29 inhibitor domain (I29I), which may act as a propeptide and prevent access of the substrate to the active site.A peptidase C1 domain (PC1) as a mature peptide was identified in the C-terminus, and six S2 subsites defined by residues Leu227, Met228, Ala294, Leu320, Gly323 and Lys370 were found within this domain.A potential N-glycosylation site located at Asn147 and the four active site residues Gln177, Cys183, His322 and Asn343 were also identified (Fig. 1b).No transmembrane region was predicted in Dd-CPL-1.The estimated MW and theoretical pI of the protein were 42.51 kDa and 6.14, respectively.

Homology modeling of Dd-CPL-1
The three-dimensional structure of Dd-CPL-1 was predicted by using the online SWISS-MODEL server with the crystal structure of human cathepsin L (PDB: 6jd8.1.A) as template.The fold of Dd-CPL-1 consisted of two domains, the L and R domains, divided by the active-site cleft, with the L domain dominated by alpha-helices and the R domain composed of alpha-helices and beta-strands (Fig. 3a).Molecular surface modelling was calculated using the PyMOL program based on the structure of Dd-CPL-1, showing the presence of charged regions and neutral regions.A hydrophobic pocket that can accommodate residues of the substrate was predicted to be located in the S2 subsites (Fig. 3b).

Expression pattern and tissue localization of Dd-cpl-1
Dd-cpl-1 was expressed at all developmental stages in D. destructor.Relatively higher expression levels were detected in J3s and female adults (Fig. 4a).Using the expression level in eggs as reference, we found that Dd-cpl-1 was upregulated 6.03-and 23.65-fold in J2, J3, respectively.Then it went down to 6.52-fold in J4 stage.Finally, in adult stage expression level of Dd-cpl-1 remained down (3.71-fold) in male adults, while it showed upregulation (up to 28.34-fold) in female adults.
In situ hybridization showed that Dd-cpl-1 was located in the bulk of eggs, juveniles and female adults (Fig. 4b-f).The digoxigenin-labelled specific probe primarily stained the esophagi and intestines in J2s (Fig. 4c), J3s (Fig. 4d), J4s (Fig. 4e) and female adults (Fig. 4f).In addition, a clear hybridization signal was also detected in the ovaries and spermathecae of female adults (Fig. 4f).No signal was observed in the control worms and eggs that were incubated with the digoxigenin-labelled sense probe (Fig. 4g-k).

Effect of Dd-cpl-1 silencing on D. destructor embryogenesis
RNAi analysis showed that the control eggs treated with dsGFP completed embryonic development from 1-cell egg to J1 and J2 stages in108 and 126 h, respectively (Fig. 5a-e), whereas the dsDd-cpl-1-treated eggs underwent abnormal development.First, approximately 60% of the dsDd-cpl-1-treated eggs that were able to developed into J2 stage showed a delayed development phenotype (Fig. 5f-j), requiring up to 120 and 144 h to reach J1 (Fig. 5i) and J2 (Fig. 5j), respectively, which was significantly delayed compared with the control eggs (P = 0.0002; Fig. 5p).Second, approximately 20% of the dsDd-cpl-1-treated eggs displayed prolonged development during early embryogenesis, arrestment at gastrula stage and could not undergo any further morphogenesis (Fig. 5k-o), which was markedly higher than that in the control group (P = 0.0005; Fig. 5q).Finally, the hatching rate of dsDdcpl-1-treated eggs was significantly lower than that in the dsGFP control group by 25.71% (P = 0.0003; Fig. 5r).The analysis of Dd-cpl-1 transcript by qPCR results showed that the expression level of Dd-cpl-1 was significantly suppressed after RNAi with dsDd-cpl-1 compared with dsGFP (P = 0.0007; Fig. 5s).

Effect of Dd-cpl-1 silencing on female fecundity and infectivity
Compared with the dsGFP control group, the average number of eggs laid by dsDd-cpl-1-treated female adults was significantly decreased by 57.43% (P = 0.01) after 8 days (Fig. 7a).Furthermore, the hatching rate of the eggs that were laid by dsDd-cpl-1-treated females was 25.68% (P = 0.0041) lower than the control group (Fig. 7b).The majority of eggs (approximately 60%) in the dsDd-cpl-1 group could not normally hatch, and eventually died.As expected, soaking female adults in dsDd-cpl for 24 h significantly diminished the expression of Dd-cpl-1 (Fig. 7c).
Our findings suggest that Dd-cpl-1 plays important roles in embryogenesis, juvenile development and female fecundity in D. destructor.We used mixed-stage nematodes to test the effect of Dd-cpl-1 silencing on infectivity.The number of nematodes colonizing the sweet potato storage roots after treatment with dsDd-cpl-1 was significantly lower than that in the dsGFP control group by 50.51% (P = 0.0002; Fig. 7d).Meanwhile, the infection area of the nematodes treated with dsDd-cpl-1 was noticeably decreased compared to the control group (P = 0.0441; Fig. 7e, f).These findings indicate that Dd-cpl-1 plays a role in D. destructor infectivity.

Discussion
Cathepsin L-like cysteine protease (CPL) is one of the most important cellular proteases and plays key roles in nematodes and many other animal parasites 18,30 .CPL genes have been identified and cloned from a few PPN species where all functional investigations were conducted using RNAi [22][23][24][25]31 . Howver, the biological functions of CPL genes in the potato rot nematode D. destructor have yet to be explored.Herein, a CPL gene, Dd-cpl-1, from D. destructor was functionally characterized.Amino acid sequence and multiple sequence alignment analyses revealed that Dd-CPL-1 exhibits domain architecture with the presence of a signal peptide followed by I29I and PC1 domains, and four conserved catalytic residues of Gln, Cys, His, and Asn, as found in other CPLs from free-living nematode 32 and insects 33 .ERFNIN and GNFD motifs located in the I29I domain are relatively conserved and they are the main signatures of CPLs 34,35 .Phylogenetic analysis indicated that Dd-CPL-1 has the closest evolutionary phylogenetic relationship with the ortholog from the free-living fungivorous nematode A. avenae sharing 75.34% amino acid sequence identity.Molecular modelling showed that the structure of Dd-CPL-1 is composed of two globular domains divided by an active-site cleft, which is quite similar to that of other nematodes, such as B. xylophilus 36 , Angiostrongylus cantonensis 37 and Trichinella spiralis 38 .These results suggest that the CPL genes are conserved across the nematode phylum and Dd-CPL-1 may share similar functions with its orthologs in other nematode species.Some previous studies have confirmed that CPL genes are essential for embryonic development in nematodes.For example, silencing of Bm-cpl genes (Bm-cpl-1, Bm-cpl-4 and Bm-cpl-5) in filarial nematode Brugia malayi resulted in malformed intrauterine embryos and abnormal embryonic viability 39 .C. elegans cpl-1 mutant embryos showed decreased cell division rate, arrest of morphogenesis and eventual death, and it was shown that Ce-CPL-1 was involved in the degradation of yolk 32,40 , which provides the major nutrients for developing embryos.Transgenic expression of the H. contortus cpl-1 or S. vulgaris cpl-1 genes rescued the embryonic lethal phenotype of the C. elegans cpl-1 mutant, supporting their function in embryonic development 41,42 .Here, we show that Dd-cpl-1 RNAi embryos of D. destructor shared similar phenotypes with the C. elegans cpl-1 mutant, such as the dramatically slow cell division, abortive morphogenesis and embryonic lethal phenotype.These results may be explained by the potential participation of Dd-CPL-1 in yolk processing in D. destructor, which resembles the function of Ce-CPL-1 in C. elegans 40 .This hypothesis requires to be demonstrated in the future.
The expression level of Dd-cpl-1 in J3s was significantly higher than that in J2s, and J4s, which indicates that CPL may be one of the enzymes required for a specific, tightly regulated process during J3 development.Cell growth and proliferation depend on the nutritional status.A programmed arrest of development, diapause has www.nature.com/scientificreports/been found at some stages of ontogenesis in many animals, and it has been shown that the diapause state may be controlled by the nutritional status 43 .The mechanistic target of rapamycin (mTOR) pathway can sense and integrate the nutritional status to regulate eukaryotic cell growth 44 .In C. elegans, TOR-deficient worms appeared to be defective in digestion or nutrient absorption, and arrested at third-stage larvae (L3) 45 .Other nutrient sensing pathways have also been identified in C. elegans, such as the Insulin/insulin-like growth factor (IGF-1) signaling (IIS) pathway 46 .IIS pathway mutants (Ce-daf-2 and Ce-age-1) arrested development in the dauer stage, an alternative third larval stage 47 .It is still unclear whether there is a dauer state in parasitic nematodes.CPLs are members of proteolytic enzymes and have been confirmed to be involved in digestion for nutrition in many animals 17,48,49 .Here we found that Dd-cpl-1 was located in the digestive system and silencing Dd-cpl-1 expression by RNAi in J3s resulted in developmental arrest in this stage.Thus, it is possible to speculate that Dd-CPL-1 may directly affect D. destructor development and growth by acting as a digestive enzyme to supply nutrition, or may play roles in the mTOR or in the IIS pathway through comparison of the phenotype (developmental arrest at third larval stage) after disruption of Dd-cpl-1, CeTOR, Ce-daf-2 and Ce-age-1 45,50,51 .Additionally, a developmental arrest phenotype in J3s was also observed after RNAi of Dd-cpl-1 at stage of J2.In contrast, when J4s were treated with Dd-cpl-1 RNAi these worms were still able to develop into adults comparable to the control nematodes.These findings suggest that diapause and other developmental decisions in D. destructor might be determined at J3 stage and during which Dd-cpl-1 functions are required, which coincide with its relatively high expression in J3s.However, the detailed regulatory mechanisms in developmental decisions remain unknown and need further study.
Theoretically, an efficient gene-silencing might result in more pronounced phenotypic changes, while a lower level of gene-silencing might lead to more subtle or intermediate phenotypes.However, it is important to note that this correlation is not always direct or predictable.The relationship between phenotype levels and silencing efficiency can be influenced by multiple variables, including the complexity of gene interactions, redundancy of biological systems, and influence of environmental factors.Our study showed that even though that the RNAi silencing efficiency of Dd-cpl-1 in J2s was comparable to that in J4s, their phenotypes were completely different, since the interfered J2s arrest at J3 stage, while the interfered J4s can develop normally into adults.On the other hand, the difference in RNAi silencing efficiency of Dd-cpl-1 in J2s and J3s is significant, but the interfered J2 and J3 animals showed a similar developmental arrest phenotype at J3 stage.
CPL genes have been reported as being implicated in parasite reproduction.Earlier studies showed that Schistosoma mansoni CPL was localized in the reproductive organs and was involved in female fecundity 52,53 .Knocking down Mi-cpl-1 gene by in vitro RNAi or plant-mediated RNAi resulted in reduced egg number of female M. incognita 23,24,31 .Recently, Bai et al. 54 reported that silencing of TsCL gene could significantly decrease newborn larvae production by T. spiralis adult females.Similarly, we found that female D. destructor with silenced expression of Dd-cpl-1 were affected in egg production.Besides, our findings also showed that the highest expression level of Dd-cpl-1 was detected in female adults and the localization of Dd-cpl-1 was observed in the ovaries and spermathecae of female adults.These results indicate that Dd-cpl-1 gene also plays an important role in the reproductive capacity of D. destructor.Intriguingly, the expression level of Dd-cpl-1 was remarkably higher in female adults than in male adults, but no noticeable change in female: male ratio after Dd-cpl-1 silencing was observed (data not shown).It is inferred that Dd-cpl-1 may perform some other sex-specific functions not yet identified.
Finally, we determined that the infectivity (colonization and infection area) of D. destructor was significantly depressed after silencing Dd-cpl-1 gene.The lower infectivity may be explained since nematodes treated with Dd-cpl-1 RNAi exhibit defects in egg viability, juvenile development, female fecundity and/or other biological processes.For example, suppression of Bm-cpl-1 elicited slow moving, incomplete and porous gut, disrupted cuticle and uncompleted molting in B. malayi 55 .Overall, our results show that reducing the expression of Ddcpl-1 gene has negative pleiotropic effect on D. destructor ontogenesis, suggesting that Dd-cpl-1 gene target for RNAi could be a good strategy for the control of the nematode.

Conclusion
In summary, we functionally characterized the CPL gene Dd-cpl-1 in D. destructor.Our results showed that Ddcpl-1 plays multiple roles throughout the life cycle of this nematode with its essential involvement in embryogenesis, juvenile development and female fecundity.Furthermore, we determined that Dd-cpl-1 indeed plays a role in nematode infectivity.Our work not only helps to understand the role of Dd-cpl-1 in D. destructor ontogenesis, but also indicates that Dd-cpl-1 could serve as a good candidate target for future RNAi-based control of D. destructor.

Figure 1 .Figure 2 .
Figure 1.Sequence analysis of Dd-cpl-1.(a) Gene structure of Dd-cpl-1.Relative positions and respective sizes of exons are indicated as boxes and introns as lines; coding sequences are indicated as black boxes; untranslated regions are indicated as white boxes.(b) Nucleotide and amino acid sequences of Dd-cpl-1.The predicted signal peptide is highlighted in yellow.The I29 inhibitor domain is highlighted in pink.The peptidase C1 domain is highlighted in blue.The potential N-glycosylation site is circled.S2 subsites are boxed.Active site residues are underlined.

Figure 3 .
Figure 3. Molecular modeling of Dd-CPL-1.(a) The predicted three-dimensional structure of Dd-CPL-1 composed of the L and R domains was made by SWISS-MODEL with human cathepsin L (PDB: 6jd8.1.A) as a template.The L and R domains are divided by an active-site cleft (indicated by the arrow).Four active site residues within the active-site cleft are highlighted in purple.(b) The electrostatic potential surface of Dd-CPL-1.Positively-and negatively-charged regions are indicated by blue and red color, respectively; the higher the charge quantity, the darker the color.Neutral regions are indicated by white color.A predicted hydrophobic pocket is indicated by the arrowhead.

Figure 4 .
Figure 4.The expression pattern and tissue localization of Dd-cpl-1 in D. destructor.(a) Temporal expression patterns of Dd-cpl-1 at different development stages.Data shown are means ± standard errors.Significant differences (P < 0.05) are indicated by different letters.(b-k) Localization of Dd-cpl-1 mRNA in D. destructor using in situ hybridization with the digoxigenin-labelled specific probe in the egg (b), second-stage juvenile (c), third-stage juvenile (d), fourth-stage juvenile (e) and female adult (f).No signal in eggs and worms were hybridized with the control sense probe (g-k).va, vulva; sa, spermatheca.

Figure 7 .
Figure 7. Effects of Dd-cpl-1 silencing on fecundity and infectivity of D. destructor.(a) Female fecundity analysis after the treatments with dsDd-cpl-1 or dsGFP.Eggs and J2s were counted after nematode pairing for eight days.Egg production means the sum of the number of eggs and J2s.(b) Hatching rates of eggs laid by female adults treated with dsDd-cpl-1 or dsGFP.Eight days after nematode pairing, the percentage of J2s was determined.(c) The expression levels of Dd-cpl-1 detected in female adults after treatments with dsDd-cpl-1 or dsGFP.(d) The colonization numbers of D. destructor in the sweet potato storage roots infected with dsDdcpl-1-or dsGFP-treated mixed-stage nematodes at 25 d post-infection.(e) The infection areas highlighted with a dashed border.(f) The infection areas indicated as the percentage value relative to the dsGFP control.Infection area was calculated by using ImageJ.Data shown are means ± standard errors from three biological replicates; each replicate contained 30 female adults for fecundity, and contained five technic replicates for infectivity.P-values were calculated by unpaired Student's t-test (*P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001).

Table 1 .
Primers used for this study.